Transcription factors represent a group of molecules within the cell that function to connect the pathways from extracellular signals to intracellular responses. Immediately after an environmental stimulus, these proteins which reside predominantly in the cytosol are translocated to the nucleus where they bind to specific DNA sequences in the promoter elements of target genes and activate the transcription of these target genes. One family of transcription factors, CCAAT/Enhancer-binding proteins (C/EBPs), regulates the expression of an extensive panel of genes that control normal tissue development and cellular function, cellular proliferation and functional differentiation. Six members of this family have been identified to date all of which form both homo- and heterodimers with other C/EBP family members as well as with members of the NFkB and Fos/Jun families of transcription factors (Lekstrom-Himes and Xanthopoulos, J. Biol. Chem., 1998, 273, 28545-28548). While all of the members of the C/EBP family have a similar modular protein structure, expression levels and tissue distributions vary widely leading to a diversity of roles (Lekstrom-Himes and Xanthopoulos, J. Biol. Chem., 1998, 273, 28545-28548).
The first member, originally isolated from soluble extracts of rat liver nuclei, is C/EBP alpha, also known as CEBPA and simply C/EBP (Graves et al., Cell, 1986, 44, 565-576; Johnson et al., Genes Dev., 1987, 1, 133-146). Studies of tissue distribution and developmental expression patterns showed that C/EBP alpha is found primarily in tissues involved in energy metabolism, with a capacity to metabolize lipids, cholesterol and other sterols (Birkenmeier et al., Genes Dev., 1989, 3, 1146-1156). The highest levels of expression were found in the liver, adipose and placental tissue, while lower levels were seen in lung and small intestine.
In studies of the rat, C/EBP alpha has been shown to be involved in the regulation of adipocyte differentiation (Samuelsson et al., Embo J., 1991, 10, 3787-3793; Shao and Lazar, J. Biol. Chem., 1997, 272, 21473-21478). Using vector-directed expression of antisens;e C/EBP RNA, the authors showed reduced expression of C/EBP alpha transcripts as well as reduced cytoplasmic triglyceride accumulation, indicating the necessity of the alpha isoform in adipocyte differentiation (Lin and Lane, Genes Dev., 1992, 6, 533-544). C/EBP alpha has also been shown to regulate chondrogenic differentiation (Vidal et al., J. Endocrinol., 1997, 155, 433-441), follicular development and ovulation (Piontkewitz et al., Dev. Biol., 1996, 179, 288-296), steroid-induced cell cylce arrest in the liver (Ramos et al., Mol. Cell. Biol., 1996, 16, 5288-5301), and controling GLUT2, a glucose transporter, promotor activity (Kim and Ahn, Biochem. J., 1998, 336, 83-90). It is also expressed in activated microglial cells after brain injury (Walton et al., Brain Res. Mol. Brain Res., 1998, 61, 11-22).
These findings are futher supported by studies in humans showing C/EBP alpha to be involved in the hormonal regulation of metabolism and in granulocyte development (Radomska et al., Mol. Cell. Biol., 1998, 18, 4301-4314; Roesler et al., J. Biol. Chem., 1998, 273, 14950-14957).
C/EBP-deficient mice have been generated for five of the six members of the C/EBP family and these have been characterized for system-specific phenotypic abnormalities. C/EBP alpha is the primary signal controlling hepatic terminal differentiation, and mice lacking C/EBP alpha have profound derangement of liver structure and function and the majority die soon after birth due to hypoglycemia. In addition, mice lacking the C/EBP alpha gene exhibit deficient granulopoiesis (Zhang et al., J. Exp. Med., 1998, 188, 1173-1184), liver development (Timchenko et al., Mol. Cell. Biol., 1997, 17, 7353-7361; Tomizawa et al., Biochem. Biophys. Res. Commun., 1998, 249, 1-5) and differentiation of myeloid precursors (Zhang et al., Proc. Natl. Acad. Sci. U. S. A., 1997, 94, 569-574).
In addition to stage-specific expression level variations, the C/EBP members also undergo multiple isoform expression arising from alternative start positions, for the alpha and beta isoforms, in 5' upstream open reading frames (Geballe and Morris, Trends Biochem. Sci., 1994, 19, 159-164; Lincoln et al., J. Biol. Chem., 1998, 273, 9552-9560). The steady-state level of the various pools of transcripts also changes as a function of age and stress challenges (Hsieh et al., Mol. Biol. Cell, 1998, 9, 1479-1494). In mice the expression of certain transcripts of one isoform has also been shown to regulate the expression of other C/EBP isoforms (Burgess-Beusse et al., Hepatology, 1999, 29, 597-601).
C/EBP alpha occurs as two isoforms in the cell, a full-length 42-kDa form and a shorter 30-kDa form. The shorter form displays alternative transactivation potential compared to the full-length protein (Lekstrom-Himes and Xanthopoulos, J. Biol. Chem., 1998, 273, 28545-28548). Disclosed in U.S. Pat. No. 5,545,563 and in the PCT application, WO 94/20113, are the DNA sequence of the entire human C/EBP alpha gene and vectors for its expression (Darlington et al., 1994; Darlington et al., 1996). Also disclosed in WO 94/20113 is the use of the C/EBP alpha gene in methods to treat cancer and other diseases. Antisense agonists of C/EBP alpha are also generally disclosed.
The pharmacological modulation of C/EBP alpha activity and/or expression may therefore be an appropriate point of therapeutic intervention in pathological conditions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of C/EBP alpha and to date, investigative strategies aimed at modulating C/EBP alpha function have involved the use of antibodies, antisense expression vectors, and gene knock-outs in mice. However, these strategies are untested as therapeutic protocols and consequently there remains a long felt need for agents capable of effectively inhibiting C/EBP alpha function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of C/EBP alpha expression.
The present invention provides compositions and methods for modulating C/EBP alpha expression, including modulation of both the long and short isoforms of C/EBP alpha.